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SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 33model of acute TB infection, making ecumicin the only TB drug lead among three known compounds targeting M. tuberculosisClpC1. Genome sequencing of spontaneous ecumicin-resistant clones and binding assays strengthened by the availability of mutated proteins implicated ClpC1 as the primary molecular target of ecumicin. Whole-genome sequencing of these isolates also revealed common mutations in ppsC and espG3. The gene ppsC encodes the phthiocerol synthesis polyketide synthase type I, PpsC, which is involved in the elongation of C22 to C24 fatty acids by the addition of malonyl-coenzyme (CoA) and methylmalonyl-CoA extender units to yield phthiocerol derivatives[11]. That ppsC is a nonessential[12%u201314] gene and that an M. tuberculosis CDC1551 ppsC transposon mutant was found to be fully sensitive to ecumicin suggest that PpsC is not likely to be the target. The gene espG3 encodes the ESX-3 secretionassociated protein EspG3 and is essential for the viability and infectivity of M. tuberculosis H37Rv [15%u201317]. While its function remains unknown, the analogous protein EspG3 in M. smegmatis is one of the core Esx-3 components that are required for both mycobactin-mediated iron acquisition and EsxG and EsxH export [18]. As ecumicin failed to protect either the wild-type or mutant EspG3 (see Fig. S2 in the supplemental material) from nonspecific proteolytic degradation, we conclude that it is unlikely that EspG3 is a target. Ecumicin%u2019s actual mode of action, stimulating its ATPase activity but blocking ClpC1-mediated protein breakdown by ClpP1P2, in other words, uncoupling ATP hydrolysis from proteolysis, is quite unusual. ClpP1P2 and its regulatory factors ClpC1 and ClpX are essential in M. tuberculosis, presumably to eliminate the critical proteins whose accumulation is toxic[10]. This bactericidal mechanism is opposite to that demonstrated for the acyldepsipeptide (ADEP) antibiotics, which nonspecifically